The present invention relates to an apparatus and scalable methods of lysing cells. The invention also relates to methods of isolating and purifying cellular components from lysed cells. The invention is particularly suited for scalable lysis of plasmid-containing bacterial cells, and subsequent preparation of large quantities of substantially purified plasmid. The resulting plasmid is suitable for a variety of uses, including but not limited to gene therapy, plasmid-mediated hormonal supplementation or other therapy, DNA vaccines, or any other application requiring substantial quantities of purified plasmid. Over the last five years, there has been an increased interest in the field of plasmid processing. The emergence of the non-viral field has caused researchers to focus on a variety of different methods of producing plasmids.
Because plasmids are large and complex macromolecules, it is not practical to produce them in large quantities through synthetic means. Instead, they must be initially produced in biological systems, and subsequently isolated and purified from those systems. In virtually all cases, biological production of plasmids takes the form of fermenting Escherichia coli (E. coli) cells containing the plasmid of interest. A number of techniques for fermenting plasmid-containing E. coli cells have been known by those skilled in the art for many years. Many fermentation processes have been published, are well known and are available in the public domain.
Cell lysis and the subsequent treatment steps used to prepare a process stream for purification are the most difficult, complex and important steps in any plasmid process. It is in this process step where yield and quality of the plasmid of interest are primarily determined for each run. The search for an optimal method, one that is continuous and truly scalable, has been an obstacle in getting acceptable processes with commercial applicability.
There are a variety of ways to lyse bacterial cells. Well-known methods used at laboratory scale for plasmid purification include enzymatic digestion (e.g. with lysozyme), heat treatment, pressure treatment, mechanical grinding, sonication, treatment with chaotropes (e.g. guanidinium isothiocyante), and treatment with organic solvents (e.g. phenol). Although these methods can be readily practiced at small scale, few have been successfully adapted for large-scale use in preparing plasmids.
Methods such as pressure treatment, mechanical grinding, or sonication can be difficult to implement at large scale. Moreover, Carlson et al. (1995, Biotechnol. Bioeng. 48, 303–315) have shown that such mechanical methods can lead to unacceptable plasmid degradation. Methods involving chaotropes and/or organic solvents are problematic to scale up because these chemicals are typically toxic, flammable, and/or explosive. Handling and disposing of such chemicals is manageable at small scale, but generally creates substantial problems at large scale. U.S. Pat. No. 6,197,553 describes a large-scale lysis technique involving treatment with lysozyme and heat. However, this technique requires carefully controlled heating and cooling of the enzymatically-treated bacterial cells to achieve lysis. The technique also has disadvantages in that it requires the use of an animal-derived enzyme (lysozyme), which can be expensive and is a potential source of biological contamination. Using animal-derived materials is quickly becoming unacceptable when preparing plasmids or other cellular components of interest for human or veterinary applications.
Currently, the preferred method for lysing bacteria for plasmid purification is through the use of alkali and detergent. This technique was originally described by Birnboim and Doly (1979, Nucleic Acids Res. 7, 1513–1523). A commonly used variation of this procedure, as described on pp. 1.38–1.39 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), is to suspend bacterial cells in 10 mL of a resuspension solution, consisting of 50 mM glucose, 25 mM Tris, pH 8.0, 10 mM EDTA. The suspension is mixed with 20 mL of a lysis solution, consisting of 0.2 N NaOH, 1% sodium dodecylsulfate (SDS) and incubated for 5–10 minutes. During this period, the cells lyse and the solution becomes highly viscous. The high pH denatures both the host genomic DNA and the plasmid DNA. The SDS forms complexes with cellular proteins, lipids, and membrane components, some of which are tightly associated with the host genomic DNA. The lysate-mixture is next treated with 15 mL of an ice-cold neutralization/precipitation solution, consisting of 3 M potassium acetate that has been adjusted to pH 5.5 with acetic acid. This acidified mixture is incubated on ice for 5–10 minutes, in part to allow plasmid DNA to renature. During this time, a white flocculent precipitate is formed. The precipitate comprises potassium SDS, which is poorly soluble under these conditions. In addition, the precipitate contains host genomic DNA, proteins, lipids, and membrane components, which remain bound to the SDS. The precipitate is subsequently removed by filtration or centrifugation, yielding a clarified lysate containing the desired plasmid, which can be subjected to various purification procedures.
This lysis method has very distinct advantages over those described above. In addition to providing efficient release of plasmid molecules from the cells, this procedure provides substantial purification of the plasmid by removing much of the host protein, lipids, and genomic DNA. Removal of genomic DNA is particularly valuable, since it can be difficult to separate it from plasmid DNA by other means. These advantages have made this a preferred method for lysing bacterial cells during plasmid purification at laboratory scale.
Unfortunately, this method presents significant challenges for scaling up. First, thorough mixing of suspended cells with lysis solution is easily managed at small scale by simply vortexing or repeatedly inverting the vessel containing the cells. However, this is impractical at large scale, where volumes may be in the range of tens or hundreds of liters. Common techniques for mixing large volumes of liquid, such as batch impeller mixing, are problematic because as some cells begin to lyse after initial mixing, they release genomic DNA that dramatically increases solution viscosity. This increase in viscosity significantly interferes with further mixing.
A second challenge is that excessive incubation at high pH after addition of alkaline lysis solution can lead to permanent denaturation of the plasmid, making it unsuitable for most subsequent uses. It is therefore necessary to ensure that the lysed cells are thoroughly mixed with neutralization/precipitation solution within a relatively narrow time frame, typically within 5–10 minutes. It is also well known that mixing at this step must be gentle (i.e. low shear). Vigorous (i.e. high shear) mixing at this step releases substantial amounts of material from the flocculent precipitate into the plasmid-containing solution. This includes large amounts of host genomic DNA and endotoxins. These substances are difficult to separate from the plasmid during subsequent purification. Thus, while complete mixing is required to precipitate all of the SDS-associated impurities and renature all of the plasmid, mixing should also be as gentle as possible. This is easily accomplished at small scale by timed addition of neutralization/precipitation solution using hand mixing techniques such as gentle swirling or inversion of the containers. In contrast, rapid yet gentle mixing is difficult to achieve at large scale. Low shear stirring or impeller mixing in batch mode requires relatively long times to achieve complete mixing, which could result in unacceptably high levels of permanently denatured plasmid. More rapid techniques such as high speed impeller mixing are likely to result in unacceptably high levels of genomic DNA and endotoxin in the plasmid-containing solution.
It has previously been believed that mixing a cell suspension and a lysis solution must be performed at very low shear. This has been particularly claimed in regard to mixing suspensions of plasmid-containing bacteria with lysis solutions comprising alkali and detergent. For example, Wan et al., in U.S. Pat. No. 5,837,529, in discussing methods of lysing plasmid-containing cells with alkali or enzymes, contend that it is crucial to handle such lysates very gently to avoid shearing genomic DNA. Similarly, Nienow et al., in U.S. Pat. No. 6,395,516, in discussing the challenges of alkaline lysis, claim that too vigorous mixing at any stage of the procedure may lead to fragmentation of genomic DNA, which may substantially contaminate the final purified product. Yet again, Bridenbaugh et al., in U.S. Patent Application No. 2002/0198372, emphasize the need for gentle mixing of cells with lysis solution. These concerns have led such investigators to develop ostensibly scalable means to gently mix suspended cells with lysis solutions. For instance, U.S. Pat. No. 5,837,529 and U.S. Patent Application No. 2002/0198372 each contemplate using static mixers to achieve continuous low shear mixing, while U.S. patent application Ser. No. 6,395,516 contemplates using a designed vessel for controlled mixing in batch mode. Such methods have clear drawbacks. In one regard, while striving to minimize excessive shear, mixing of the cell suspension with the lysis solution may be incomplete. In another regard, using static mixers limits process flexibility. As described in U.S. Patent No. 2002/0198372, it is necessary to optimize the number of static mixing elements, as well as the flow rates of the fluids passing through the elements. Such optimization restricts the amount of material that may be processed in a given time with the optimized static mixing apparatus. This limits the ability to increase process scale, unless a new, higher-capacity static mixing apparatus is constructed and optimized. Use of batch mixing vessels, as described in U.S. Pat. No. 6,395,516, has comparable drawbacks. Achieving complete mixing in all regions of a batch mixing vessel is well known by those of skill in the art to be challenging. Furthermore, batch mixing vessels are poorly suited for applications that require a controlled exposure time wherein the cell suspension is contacted with the lysis solution. In particular, it is well known that prolonged exposure of plasmid-containing cells to alkali may lead to the formation of excessive amounts of permanently denatured plasmid, which is generally inactive, undesirable, and difficult to subsequently separate from biologically active plasmid. Typically, it is desirable to limit such exposure times to about 10 minutes or less. Achieving such limited exposure times is difficult or impossible using large scale batch mixing.
Removal of the flocculent precipitate is yet another challenge in scaling up alkaline lysis. Complete removal is desirable to eliminate the genomic DNA and other impurities trapped in the precipitate. At the same time, the precipitate must not be subjected to excessive shear. Otherwise, large amounts of genomic DNA, endotoxins, and other impurities are released from the precipitate and contaminate the plasmid-containing solution. At laboratory scale, the precipitate is readily removed by simple filtration, batch centrifugation, or both. However, batch centrifugation is highly impractical at large scale. Continuous centrifugation at large scale is also unsuitable because it subjects the precipitate to high shear stress, releasing unacceptable levels of impurities. Filtration at large scale is problematic due to the somewhat gelatinous, cheese-like consistency of the precipitate, which readily clogs even depth or bag filters.
Notwithstanding the above challenges, a variety of investigators have developed claimed improvements of the alkaline lysis method, or otherwise attempted to adapt it into a scalable production process. Kresheck and Altschuler, in U.S. Pat. No. 5,625,053, describe the use of non-ionic alkyldimethylphosphine oxide detergents in place of SDS. Use of these detergents is claimed to offer certain advantages relevant to large-scale preparation of pharmaceutical grade plasmid. However, the claimed improvements do not address the scalability issues described above.
Thatcher et al., in U.S. Pat. No. 5,981,735, describe a modification where the amount of NaOH added to the suspended cells is carefully controlled to ensure that the pH remains approximately 0.1 pH units below the point that results in substantial permanent denaturation of plasmid. This approach may address the issue of time-dependent generation of permanently denatured plasmid, but requires very precise pH control, which can be difficult at large scale. Furthermore, the preferred pH level must be determined in advance for each plasmid and host cell combination. Most importantly, this approach does not address the challenges of handling and mixing large liquid volumes.
Wan et al., in U.S. Pat. No. 5,837,529, describe a process of lysing cells, comprising the use of static mixers to mix suspended cells with a lysis solution (e.g. 0.2 N NaOH, 1% SDS), as well as to mix lysed cells with a precipitating solution (e.g. 3 M potassium acetate, pH 5.5). Static mixers are claimed to be particularly advantageous by providing a high degree of mixing at a relatively low shear, and are also amenable to a continuous flow-through process. A similar process using static mixers is described by Bridenbaugh et al. in WO 00/05358. Such procedures offer certain advantages, but drawbacks remain. As shown in WO 00/05358, both the number of static mixing elements and the solution linear flow rates must be carefully controlled at each stage. Using too few mixing elements or a low linear flow rate leads to inadequate mixing and poor plasmid yields. Using too many elements or a high linear flow rate leads to excessive shearing and release of genomic DNA into solution. These parameters must be experimentally optimized, and any efforts to increase process scale require re-optimization of element number and flow rate, limiting process flexibility and the robustness of this method for routine use.
Marquet et al. (1995, Biopharm 8, 26–37) describe the use of batch mixers originally designed for use in the food industry. They claim that these mixers can provide thorough mixing at low shear rates, making them suitable for use during large-scale alkaline lysis of plasmid-containing cells. However, batch mixing of large fluid volumes in tanks is often very difficult to scale up, particularly when there are dramatic differences in fluid viscosity, or when mixing itself leads to dramatic increases in viscosity. Batch mixing is also problematic when coupled with short, time-sensitive incubation steps. All of these concerns pertain to alkaline lysis, making batch mixing particularly unsuitable.
Thus, despite the efforts of previous investigators, there is still a clear need for new and improved procedures to perform alkaline lysis at large scale. A preferred process would address a series of key challenges, including: (1) thorough, rapid, and robust mixing of cells and lysis solution, to efficiently lyse cells and release plasmid; (2) time-controlled incubation of lysed cells in alkali, to prevent permanent plasmid denaturation; (3) thorough, rapid, and gentle mixing of alkaline lysate with neutralization/precipitation solution, to efficiently precipitate contaminating cellular components without releasing excess genomic DNA and endotoxin into the plasmid-containing solution; and (4) efficient yet gentle removal of the flocculent precipitate, again without releasing excess genomic DNA and endotoxin into the plasmid-containing solution. Furthermore, such a preferred process would be readily scalable, robust, suitable for use in all applications, would contain no animal derived products, and would be cost effective.
There is also a need for improved procedures for purifying plasmids from large-scale microbial cell lysates. In particular, the emerging fields of non-viral gene therapy, plasmid-mediated therapy and DNA vaccines require gram or even kilogram amounts of purified plasmid suitable for pharmaceutical use. It is thus necessary to purify plasmids away from the primary impurities remaining in the lysate, including residual genomic DNA, RNA, protein, and endotoxin. An ideal process should provide substantially pure material in high yield, be easy to scale up, involve a minimal number of steps, and be simple and inexpensive to perform. Any use of enzymes or animal-derived products should be avoided, as such reagents tend to be expensive and more importantly, are potential sources of contamination. Similarly, use of alcohols and organic solvents is to be avoided, as they are generally toxic, flammable, explosive, and difficult to dispose of in large quantities. Known or suspected toxic, mutagenic, carcinogenic, teratogenic, or otherwise harmful compounds should not be used. Finally, the process should avoid the need for expensive equipment such as large scale or continuous centrifuges, or gradient producing chromatography skids.
Various attempts have been made to develop a plasmid purification process that meets these ideals. For example, Horn et al., in U.S. Pat. No. 5,707,812, describe an integrated process involving alkaline lysis, filtration with diatomaceous earth, concentration and desalting by ultrafiltration/diafiltration (UF/DF), overnight precipitation of plasmid with 8% polyethylene glycol (PEG) 8000, centrifugation, resuspension, precipitation of impurities with 2.5 M ammonium acetate, centrifugation, precipitation of plasmid with isopropanol, centrifugation, resuspension, anion exchange column chromatography in the presence of 1% PEG 8000 on Q Sepharose™ (Amersham Biosciences Corp., Piscataway, N.J.) with step elution, plasmid precipitation with isopropanol, centrifugation, resuspension, and gel filtration column chromatography on Sephacryl™ S-1000 (Amersham Biosciences Corp., Piscataway, N.J.). Plasmid yields, quality, and purity were not described. Similar processes are disclosed by Marquet et al. in U.S. Pat. No. 5,561,064. These processes are not easily scaled, due to the multiple plasmid precipitations and centrifugations. In addition, achieving adequate resolution with gel filtration column chromatography typically requires relatively large columns. Use of isopropanol in multiple steps is another disadvantage of this process.
U.S. Pat. No. 5,990,301, issued to Colpan et al., describes an integrated process involving alkaline lysis, clarification by centrifugation and filtration, incubation with salt (NaCl) and nonionic detergent, anion exchange by DEAE column chromatography, isopropanol precipitation, centrifugation, and resuspension. The resulting plasmid was reported to contain “no detectable” RNA, genomic DNA, or endotoxin, but detection methods and limits were not described. This process has numerous scalability issues. DEAE resins typically have relatively low capacity for plasmid. Furthermore, using isopropanol precipitation and centrifugation for product concentration and desalting is not feasible at large scale.
U.S. Pat. No. 6,197,553, issued to Lee and Sagar, describes an integrated process involving cell wall digestion with lysozyme, lysis by passing through a flow-through heat exchanger to heat the cell suspension to about 80° C., clarification by centrifugation, diafiltration, treatment with RNase, diafiltration, anion exchange column chromatography on POROS® PI/M (Applied Biosystems, Foster City, Calif.) with NaCl gradient elution, reverse phase chromatography on POROS® R2/M with isopropanol gradient elution, and UF/DF. Final product contained 2.9% genomic DNA, <1% protein, <1% RNA, and endotoxin levels of 2.8 endotoxin units (EU) per milligram of plasmid. However, this process suffers from the use of two enzymes (lysozyme and RNase), gradient-based anion exchange chromatography, and gradient-based reverse phase chromatography using isopropanol. These present substantial scalability and/or regulatory issues.
U.S. Pat. No. 6,410,274, issued to Bhikhabhai, describes a process involving alkaline lysis, filtration, precipitation of RNA and genomic DNA with CaCl2, centrifugation, filtration, anion exchange column chromatography on Q Sepharose™ XL (Amersham Biosciences Corp., Piscataway, N.J.) with step elution, and further anion exchange column chromatography on Source™ 15Q (Amersham Biosciences Corp., Piscataway, N.J.) with step elution. Final product was reported to contain 0.6% genomic DNA (by PCR), 100% supercoiled plasmid (by anion exchange high performance liquid chromatography, “HPLC”), and no detectable RNA (by reverse phase HPLC), protein (by Micro BCA™ assay, Pierce Biotechnology, Rockford, Ill.), or endotoxin (by limulus amebocyte lysate, “LAL”). The use of two successive anion exchange steps is an obvious inefficiency of this process. Furthermore, the process relies on column chromatographic techniques, which involve expensive hardware and resins.
WO 00/05358, submitted by Bridenbaugh et al., describes a process where plasmid-containing cells are resuspended in the presence of RNase. A continuous lysis procedure is described, where the resuspended cells and an alkaline lysis solution are simultaneously pumped through a static mixer to achieve lysis. The lysate is then mixed with potassium acetate precipitation solution via a second static mixer. The precipitated lysate then passes into a continuous centrifuge to remove the flocculent precipitate, resulting in a clarified lysate. Clarified lysate is filtered to remove fine particulates and purified by anion exchange column chromatography using Fractogel® TMAE-650M (Merck KGaA, Darmstadt, Germany). The anion exchange eluate is then passed through glass and nylon filters, which are claimed to help remove endotoxin and genomic DNA. Purified plasmid was then concentrated and desalted by UF/DF, and sterilized by filtration. Final endotoxin levels were 16.2 EU/mg. Residual RNA, protein, and genomic DNA were said to routinely be <2%, <0.1%, and <1%, respectively. Use of continuous centrifugation is a significant drawback of this process, due to high shear rates and subsequent release of excess genomic DNA into solution, as well as the high cost of such equipment. Use of RNase is a further drawback of this process from a regulatory standpoint.
U.S. Patent Application No. 2001/0034435, submitted by Nochumson et al., describes a process where plasmid-containing cells are lysed with alkali and SDS in a continuous process using static mixers. The lysate is neutralized by continuous addition (via a second set of static mixers) of a neutralization/precipitation solution. The neutralized lysate is held for 6–12 hours at 4° C. to precipitate the majority of the RNA. The flocculent precipitate and the precipitated RNA are removed by centrifugation and/or filtration, and the plasmid-containing solution is subjected to anion exchange column chromatography using Fractogel® TMAE-650S (Merck KGaA, Darmstadt, Germany). Plasmid is then eluted and subjected to hydrophobic interaction chromatography (“HIC”), also in column format, using Octyl Sepharose™ 4FF (Amersham Biosciences Corp., Piscataway, N.J.). Under appropriate conditions, genomic DNA, RNA, and endotoxin bind to the resin, while plasmid passes through. After HIC, the product is concentrated and desalted by UF/DF, and sterile filtered. Detailed information on yields and purity were not described in this application. However, plasmid binding capacities for the resins are relatively low (1–3 mg/mL for the anion exchange, and <1 mg/mL for the HIC if used in binding mode), and again, there is a reliance on column chromatography.
Varley et al. (1999, Bioseparation 8, 209–217) describe a process consisting of optimized alkaline lysis with RNase treatment, bag depth filtration, expanded bed anion exchange chromatography, ultrafiltration, and size exclusion chromatography. Similar processes are disclosed in U.S. Pat. No. 5,981,735 by Thatcher et al. In these processes, the pH during alkaline lysis was carefully controlled at a point just below the empirically determined level that leads to permanent plasmid denaturation. The investigators claim that this allows extended incubation in alkali, presumably to maximize lysis and/or to degrade RNA without damaging plasmid. Impurities were reported to be <2% genomic DNA (by PCR), 0.2% RNA (by HPLC), <0.1% protein, and 2.5 EU/mg endotoxin. However, the process contains several undesirable elements, including use of RNase, bag depth filtration, column-based anion exchange, and size exclusion chromatography. Performing the controlled alkaline lysis requires carefully determining the ideal pH for a given combination of host, plasmid, and growth conditions, suggesting that this step may not be very robust.
As the above examples suggest, column chromatography is often a preferred element in plasmid purification. Anion exchange chromatography is well suited for separating plasmids from certain impurities such as proteins, because plasmids, like all nucleic acids, have a high negative charge density. Thus, many known plasmid purification processes include an anion exchange step. However, anion exchange chromatography is less suited for separating plasmids from other nucleic acids with similar negative charge densities, such as genomic DNA or RNA. Thus, anion exchange chromatography is frequently combined with another chromatographic step to achieve sufficiently pure plasmid. As discussed above, these may include size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography, and even additional anion exchange chromatography. Other chromatographic techniques are also known. For example, Wils and Ollivier, in WO 97/35002, disclose methods for purifying plasmids with ceramic hydroxyapatite. Comparable methods are disclosed by Yamamoto in U.S. Pat. No. 5,843,731. Ion-pair or matched ion chromatography may be used, as disclosed, for example, by Gjerde et al. in U.S. Pat. No. 5,986,085. Silica, glass beads, or glass fibers may also be used, as disclosed, for example, by Padhye et al. in U.S. Pat. No. 5,808,041, by Woodard et al. in U.S. Pat. No. 5,650,506, and by Woodard et al. in U.S. Pat. No. 5,693,785. Alternatively, magnetic beads or particles may be used, as disclosed, for example, by Reeve and Robinson in U.S. Pat. No. 5,665,554, and by Hawkins in U.S. Pat. No. 5,898,071. Affinity methods are also known, with examples being disclosed by Ji and Smith in U.S. Pat. No. 5,591,841, and by Cantor et al. in U.S. Pat. No. 5,482,836.
Despite the frequent use of column chromatography, there remain substantial limitations to this general technique. Chromatography resins are often expensive, and must be carefully packed into specially designed column hardware. Reproducibly packing large-scale chromatography columns is a significant challenge, as discussed by Rathore et al. (2003, Biopharm International, March, 30–40). Furthermore, in regards to plasmids, traditional chromatography resins typically offer relatively low binding capacities. For example, Levy et al. (2000, Trends Biotechnol. 18, 296–305) examined a variety of commercially available anion exchange resins and found that all exhibited plasmid binding capacities of about 5 mg/mL or less, with most exhibiting capacities of about 2 mg/mL or less. Moreover, accessibility to binding sites for large molecules like plasmids is mostly by diffusion and resins have a limited pressure drop resulting in low throughput, making these steps time consuming, costly and impractical.
Thus, it is desirable to develop a purification process that retains the advantages of column chromatography while avoiding its drawbacks. Use of membrane chromatography offers a potential solution. Membrane-based techniques typically offer substantially higher binding capacities, as well as very high flow rates. Expensive large-scale column hardware is not required. In addition, the difficulties associated with column packing are avoided, as well as the need for costly cleaning validation studies.
Certain previous investigators have disclosed membrane-based methods for purifying plasmids. For instance, Nieuwkerk et al., in U.S. Pat. No. 5,438,128, describe the use of an assembly containing a plurality of stacked microporous anion exchange membranes for purifying nucleic acids, including plasmids. However, their method is described for relatively small-scale purification of up to several hundred micrograms of plasmid. Furthermore, although the purified plasmid was stated to be RNA and protein free, there was no disclosure that the provided methods could substantially eliminate genomic DNA or endotoxin. Demmer and Nussbaumer, in U.S. Pat. No. 6,235,892, disclose a method of purifying nucleic acids, including plasmids, from a solution containing endotoxin, using a microporous weakly basic anion exchange membrane. Similarly, in WO 01/94573, Yang et al. claim a process involving two (or more) separate membranes, wherein one binds plasmid and the second binds endotoxin. The investigators state that their methods provide plasmid that is suitable for use in many pharmaceutical applications, but no data is provided to support this statement.
Thus, none of the disclosed membrane-based purification processes is demonstrably adequate for preparing substantially pure plasmid that is acceptable for pharmaceutical, veterinary, or agricultural applications. There is therefore a need for a purification process that employs membrane-based chromatographic separations, avoids column chromatography, and provides substantially pure plasmids or other biologically active molecules of interest.